Although very useful for treating heart failure, digitalis is highly toxic; this toxicity is believed to be related to the drug's inhibition of the NaK-pump. Our final aim in these studies is to clarify and thereby eliminate or reduce the toxicity of digitalis. Our previous studies using the fragmental NaK-ATPase preparations have shown that this drug binds with special NaK-ATPase conformers that are formed during phosphorylation of the enzyme. The interconversion of these conformers during ATO hydrolysis has been studied extensively in the fragmental preparation, and the sidedness effects of various ligands on the conformers' specific sites have been recently clarified. We next plan to look at how the NaK-pump is affected by an alkali ion or a proton gradient across the membrane bilayer and how the sidedness effects of ions affect the cardiac glycoside-NaK-pump interaction. Using our improved NaK-ATPase proteoliposomes, we plan to carry out the following studies: (a) A study of the effects of imposed alkali ion and proton gradients on the phosphorylated intermediates of NaK-ATPase reaction, on Na+ and K+ transport, and on the ADP-ATP exchange in proteoliposomes. These studies are important for understanding the role of electrochemical potentials in the proteoliposomal NaK-ATPase. The anion effects will also be studied. Our main focus will be conversion of the ADP-sensitive, K+-sensitive phosphorylated form (E-1P) into the ADP-insensitive, K+-sensitive form (E-2P) in the NaK-pump with special emphasis on E-P, the proposed intermediate in the E-1P leads to E-2P conversion. (b) A Study of the association and dissociation rates of membrane-permeable cardiac glycosides (e.g., 4'-acetyl digitoxigenin monodigitoxide) and of fluorescent derivatives of cardiac glycosides with the NaK-ATPase proteoliposomes in the presence of various ligands and ionophores. These studies using NaK-ATPase proteoliposomes should resolve many disagreements on the cardiac glycoside-NaK-pump interaction, and we hope to find the clue to reducing the toxicity of digitalis.